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<p>Replication timing (RT), the temporal order in which genomic regions replicate, is considered a functional feature of multiple cellular processes and chromatin organization. Two approaches to measure RT are the Repli-seq and DNA copy number (also called S/G1) methods. We previously adapted Repli-seq using 5-ethynyl-2’- deoxyuridine (EdU) pulse-labeling and bivariate flow sorting, and while the approach offers high resolution and exposes heterogeneity in timing, the S/G1 method is a simpler, faster and less resource-intensive assessment. Here we modified the S/G1 technique by using EdU labeling (EdU-S/G1) to facilitate better separation of replicating from non-replicating nuclei during flow sorting, which enables the collection of a more pure sample of G1-phase nuclei. When comparing the three methods we found that profiles from the S/G1 and EdU-S/G1 methods are highly correlated with each other and with Repli-seq profiles for early replication. We also found that the EdU-S/G1 approach offers a better representation of replication in early and late S phase than the conventional S/G1 method. However, the high reproducibility of RT profiles among all three methods indicates that considerations of cost and sample availability can drive the decision of which method to choose.</p>

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A comparison of genomic methods to assess DNA replication timing

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年5月22日

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  • Emily Wheeler,
  • Leigh Mickelson-Young,
  • Emily E. Wear,
  • Mason Burroughs,
  • Hank W. Bass,
  • Lorenzo Concia,
  • William F. Thompson,
  • Linda Hanley-Bowdoin

摘要

Replication timing (RT), the temporal order in which genomic regions replicate, is considered a functional feature of multiple cellular processes and chromatin organization. Two approaches to measure RT are the Repli-seq and DNA copy number (also called S/G1) methods. We previously adapted Repli-seq using 5-ethynyl-2’- deoxyuridine (EdU) pulse-labeling and bivariate flow sorting, and while the approach offers high resolution and exposes heterogeneity in timing, the S/G1 method is a simpler, faster and less resource-intensive assessment. Here we modified the S/G1 technique by using EdU labeling (EdU-S/G1) to facilitate better separation of replicating from non-replicating nuclei during flow sorting, which enables the collection of a more pure sample of G1-phase nuclei. When comparing the three methods we found that profiles from the S/G1 and EdU-S/G1 methods are highly correlated with each other and with Repli-seq profiles for early replication. We also found that the EdU-S/G1 approach offers a better representation of replication in early and late S phase than the conventional S/G1 method. However, the high reproducibility of RT profiles among all three methods indicates that considerations of cost and sample availability can drive the decision of which method to choose.